INSIGHTS on Microscopy & Imaging: Probe-based Techniques

In contrast to radiation-based optical and electron microscopy, scanning probe microscopy (SPM) employs an atomically fine mechanical probe that scans across a surface and moves along the z-axis under the influence of weak atomic forces on the sample or the tunneling force of electrons emitted from the tip. The two main scanning probe techniques are atomic force microscopy (AFM) and scanning tunneling microscopy (STM). Both have many variants, but all share the common characteristic of creating a 3-D surface map based on topography, electrical modulus, elasticity modulus, contact, strain, density, and many others.

AFM Semiconductor Characterization Solution Dimension Icon® SSRM-HR / Bruker / www.bruker.comThe downside to SPM methods is they are limited to very small sample regions and cannot tolerate deep topography like EM can. The “images” consist of 3-D mesh representations that appear more like computer-generated mesh maps than photographs. “SPM images are less easily interpretable by laymen than SEM images are, but they contain information that is extremely valuable,” notes Vern Robertson.

AFM is a high-resolution imaging technique that enables observation and manipulation of molecular- and atomic-level features. AFM functions by bringing a cantilever tip into contact with the sample surface. An ionic repulsive force from the surface applied to the tip bends the cantilever upward. The amount of bending, measured by a laser spot reflected onto a split photodetector, is used to calculate a force. By keeping force constant during scanning, the tip’s vertical movement follows the surface profile and is recorded as the surface topography.

“The ability to make in situ observations, to image events occurring in fluids, and to precisely control the temperature and environmental conditions under which a sample is imaged enhances the utility of AFM for leading-edge research,” says Jing-Jiang Yu, application scientist at Agilent Technologies.

Because AFM does not depend on light, resolution is defined not by the diffraction limit but by the shape and dimensions of the probing tip— usually a few nanometers in radius. “That’s why AFM achieves submolecular resolution, down to the angstrom level for hard samples, and provides 3-D images,” says Andrea Slade, PhD, life science applications scientist at Bruker Nano Services (Santa Barbara, CA).

A misconception regarding fluorescence microscopy is that it achieves molecular resolution. What it actually does is rely on a reporter molecule to locate details of interest—single molecules or collections of molecules. “With AFM you’re actually seeing the molecule without any sort of labeling,” Slade adds.

AFM works as well in fluids as it does in air, which is a boon to the life sciences. The highest-resolution AFM requires attachment, but some movement is tolerated. The technique works for characterizing cell dynamics including motion, protein interactions, the effects of proteins or peptides on bacterial cells, and many other dynamic microscopy applications where EM and optical techniques are lacking.

As a mechanical method, AFM measures forces and force-activated processes such as cell pores opening and closing or higher-order changes in protein structure. Some groups are investigating AFM for medical diagnoses based on its ability to measure nano-elastic modulus differences between healthy and diseased cells. One Bruker customer has mapped flexibility versus stiffness across the surface of a single molecule of the protein bacteriorhodopsin, a membrane protein responsible for vision.

The major drawback is that AFM, as a rasterscanning technique, is significantly slower than most other types of microscopy. Fixed-sample analysis with AFM takes several minutes, which stretches to an hour or more for live cells. By the time the AFM device acquires one segment of the image, other parts may have become irrelevant due to some change. Bruker has developed a fast-scan AFM system that allows image acquisition on a time scale that matches secondary microscopy more closely.

According to Slade, many biologists are still unfamiliar with AFM. “They’ve been limited by optical microscopy but believed that AFM was too slow.” Fast-scan AFM can image cell migration, the rearrangement of the actin cytoskeleton, and membrane events that are near and dear to cell biologists without the restraint of the diffraction limit. “Now they’re actually able to see those processes.”

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